About

Daguan Nong is an Assistant Research Professor in the Department of Biomedical Engineering at Penn State University. His research focuses on the molecular mechanisms of enzymatic activity related to cellulose degradation, including the inhibition of cellulase enzymes by products such as cellobiose. His work involves advanced imaging techniques, such as multi-wavelength microscopy combining TIRFM and IRM modalities, to investigate the dynamics of processive enzymes at the single-molecule level. He has contributed to understanding how lignin impairs cellulase activity by impeding enzyme movement, and his research extends to studying vesicle transport efficiency through kinesin motor clustering. Nong's research aims to elucidate the fundamental biological processes involved in biomass degradation and enzyme function, with implications for biofuel production and biotechnological applications.

Research topics

• Chemistry
• Biochemistry
• Organic chemistry
• Materials science
• Nanotechnology
• Physics
• Biophysics
• Optics
• Biology

Selected publications

• The Carbohydrate Binding Module of TrCel7A Aids in Navigating the Complexity of Plant Cell Walls

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-09-18

  preprint

  Abstract Efficient enzymatic deconstruction of plant cell walls is critical for utilization of lignocellulose biomass. Key enzymes in this process are cellobiohydrolases, a class of cellulases that processively degrade crystalline cellulose. Many cellobiohydrolases possess a carbohydrate-binding module (CBM), yet the importance of CBMs in substrate interaction remains unclear. Here, we use single-molecule fluorescence microscopy to investigate how CBM1 of Trichoderma reesei Cel7A influences enzyme binding and motility on cellulose substrates of varying complexity. We compare wild-type Cel7A with a truncated variant lacking CBM1 (Cel7A ΔCBM ) on bacterial cellulose (BC), phosphoric acid swollen cellulose (PASC), delignified milkweed cellulose (MWC), and holocellulose nanofibrils (hCNF). While both variants showed similar steady-state binding densities on BC and PASC, Cel7A ΔCBM exhibited reduced binding on MWC and hCNF, with the greatest reduction on the hemicellulose-rich hCNF. Alkali removal of hemicellulose partially restored Cel7A ΔCBM binding, suggesting a role for CBM1 in substrate navigation and productive binding sites recognition. Kinetic analyses revealed that CBM1 enables a rapid binding mode absent in the truncated variant. Comparisons with isolated CBM3 further showed that CBMs are capable of fast substrate association. These findings demonstrate that CBMs enhance cellulase-substrate interactions by accelerating binding, enabling navigation of the complex environment of plant cell walls. Our results emphasize the importance of CBMs in natural cellobiohydrolase function and highlight their value in the design of improved cellulases for industrial biomass conversion.

  PublisherDOI

• BPS2025 - Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose

  Biophysical Journal · 2025-02-01

  article1st authorCorresponding
  PublisherDOI

• Motor clustering enhances kinesin-driven vesicle transport

  Biophysical Journal · 2025-05-05 · 2 citations

  article
  PublisherDOI

• Motor Clustering Enhances Kinesin-driven Vesicle Transport

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-10-27 · 1 citations

  preprintOpen access

  Abstract Intracellular vesicles are typically transported by a small number of kinesin and dynein motors. However, the slow microtubule binding rate of kinesin-1 observed in in vitro biophysical studies suggests that long-range transport may require a high number of motors. To address the discrepancy in motor requirements between in vivo and in vitro studies, we reconstituted motility of 120-nm-diameter liposomes driven by multiple GFP-labeled kinesin-1 motors. Consistent with predictions based on previous binding rate measurements, we found that long-distance transport requires a high number of kinesin-1 motors. We hypothesized that this discrepancy from in vivo observations may arise from differences in motor organization and tested whether motor clustering can enhance transport efficiency using a DNA scaffold. Clustering just three motors improved liposome travel distances across a wide range of motor numbers. Our findings demonstrate that, independent of motor number, the arrangement of motors on a vesicle regulates transport distance, suggesting that differences in motor organization may explain the disparity between in vivo and in vitro motor requirements for long-range transport. Significance Statement Intracellular vesicles frequently travel long distances, despite having few kinesin and dynein motors. By reconstituting liposome motility with kinesin-1 motors, we demonstrate the need for high motor copy numbers for long-range transport when motors are randomly distributed on the liposome surface. We further show that motor clustering reduces the required motor number, emphasizing its potential role in enhancing transport efficiency. Our findings highlight the significance of motor organization in regulating intracellular transport and suggest that motor clustering, such as by scaffolding proteins or lipid domains, influences bidirectional transport outcomes.

  PublisherOA PDFDOI

• Lignin impairs Cel7A degradation of in vitro lignified cellulose by impeding enzyme movement and not by acting as a sink

  Biotechnology for Biofuels and Bioproducts · 2024 · 18 citations

  • Chemistry
  • Biochemistry
  • Organic chemistry

  BACKGROUND: Cellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink. RESULTS: We used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei, as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs. CONCLUSIONS: In an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.

  PublisherOA PDFDOI

• Author response for "Xylan inhibition of cellulase binding and processivity observed at single-molecule resolution"

  2024-02-29

  peer-review
  PublisherDOI

• Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose

  Proceedings of the National Academy of Sciences · 2024-04-22 · 12 citations

  articleOpen access1st author

  Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a K i of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme’s velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the “back door” product release site to slow activity and to the “front door” substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.

  PublisherOA PDFDOI

• High-speed tracking of KIF1A motors by interferometric scattering microscopy

  Biophysical Journal · 2024-02-01

  article1st authorCorresponding
  PublisherDOI

• Xylan inhibition of cellulase binding and processivity observed at single-molecule resolution

  RSC Sustainability · 2024-01-01 · 2 citations

  articleOpen access

  Efficient cellulose degradation by cellulase enzymes is crucial for using lignocellulosic biomass in bioenergy production. Single-molecule microscopy showed that xylan hinders the efficiency of cellulase by inhibiting its binding to cellulose and impeding the processivity of bound enzyme molecules.

  PublisherOA PDFDOI

• Xylan inhibition of cellulase binding and processivity observed at single-molecule resolution

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-01-29

  preprintOpen access

  Abstract Efficient cellulose degradation by cellulase enzymes is crucial for using lignocellulosic biomass in bioenergy production. In the cell wall of plants, cellulose is bound by lignin and hemicellulose, which are key factors contributing to the recalcitrance of plant biomass. These non-cellulosic cell wall components are known to interfere with the function of cellulolytic enzymes. While the effects of lignin have been studied extensively, the contribution of xylan, the major hemicellulose in the secondary cell walls of plants, is often overlooked. To study those effects, we generated model cell wall composites by growing bacterial cellulose supplemented with varying concentrations of purified xylan. We used single-molecule microscopy to image and track fluorescently labeled Tr Cel7A, a commonly used model cellulase, as it binds and hydrolyses cellulose in these synthetic composites. We found that minute amounts of xylan are sufficient to significantly inhibit the binding of Cel7A to cellulose. The inclusion of xylan also reduced considerably the proportion of moving enzyme molecules, without affecting their velocity and run length. We suggest that, when available at low concentrations, xylan thinly coats cellulose fibrils, and incorporates as continuous patches when available at higher concentrations. Non-productive binding of Cel7A to xylan was not found to be a major inhibition mechanism. Our results highlight the importance of targeting xylan removal during biomass processing and demonstrate the potential of using single-molecule imagining to study the activity and limitations of cellulolytic enzymes. Graphical abstract

  PublisherOA PDFDOI

Frequent coauthors

• William O. Hancock

  Pennsylvania State University

  16 shared

• Zachary K. Haviland

  Pennsylvania State University

  13 shared

• Charles T. Anderson

  Pennsylvania State University

  13 shared

• Ming Tien

  Pennsylvania State University

  11 shared

• Kate L. Vasquez Kuntz

  Pennsylvania State University

  6 shared

• Nerya Zexer

  Pennsylvania State University

  6 shared

• Zhenyu Qian

  Shanghai University of Sport

  4 shared

• Guanghong Wei

  Fudan University

  4 shared

Labs

• Biomedical EngineeringPI